NONENZYMATIC GLYCATION OF GLOMERULAR BASEMENT MEMBRANE

慢性肾功能衰竭的病因有哪些？ 健康问题。 糖尿病是肾脏疾病的最大单一原因。 疾病 拟议研究的长期目标是促进 在了解糖尿病肾病的发病机制方面， 分子水平。 在糖尿病中，高血糖影响许多代谢 这些变化中的每一种都可能有助于发展 糖尿病并发症 其中一个主要的改变是非酶促的 蛋白质的糖基化。 该过程分两步进行：第一步， 第一种是可逆的（形成Amadori产物），第二种是不可逆的 一个（形成交联产物）。 由于它们的半衰期很长， 基底膜大分子是该过程的主要目标。 在 糖尿病肾病，最突出的改变之一是 肾小球基底膜增厚和通透性增加。 因此，必须了解非酶促反应的影响， 肾小球基底膜及其组分上的糖基化。 我们 本实验首先分离小牛肾小球基底膜， 它们在葡萄糖的存在下，以促进它们的非酶促 葡萄糖基化;这些孵育将在不存在或不存在葡萄糖基化的情况下进行。 存在氨基胍，一种已知抑制交联的物质 阵 第二，检查葡萄糖掺入的程度， 交联形成和葡萄糖基化诱导的结构改变 完整的肾小球基底膜，使用分光光度计和高 分辨率扫描电子显微镜技术 第三，提取 肾小球基底膜的三种主要大分子成分 (type IV胶原蛋白、层粘连蛋白和巢蛋白/巢蛋白）， 凝胶渗透和离子交换色谱法，并确定可能的 使用凝胶电泳的葡糖基化诱导的结构改变， 放射自显影和旋转阴影电子显微镜。 四是大力 检查提取的大分子与每一种相互作用的能力， 其他和定义葡萄糖基化诱导的缺陷相互作用， 比浊法、固相结合测定法和旋转阴影电子 显微镜 这些研究将拓宽我们对这些机制的认识 肾小球基底的潜在结构和功能改变 膜在糖尿病，并希望将导致旨在 减少糖尿病并发症。

Renal disease leading to chronic renal failure represents a severe public health problem. Diabetes mellitus is the largest single cause of renal disease. The long term objective of the proposed studies is to contribute in the understanding of the pathogenesis of diabetic kidney disease at the molecular level. In diabetes, hyperglycemia affects many metabolic pathways and each of these changes may contribute to the development of diabetic complications. One of the major alterations is the nonenzymatic glucosylation of proteins. This process occurs in two steps: a first, reversible one (formation of Amadori products) and a second, irreversible one (formation of crosslinked products). Because of their long half-life, basement membrane macromolecules are primary targets of this process. In diabetic kidney disease, one of the most prominent alterations is the thickening and increased permeability of the glomerular basement membrane. Therefore, it is imperative to understand the effect of nonenzymatic glucosylation on the glomerular basement membrane and its components. We propose: First, to isolate calf glomerular basement membranes and incubate them in the presence of glucose in order to promote their nonenzymatic glucosylation; these incubations will be performed in the absence or the presence of aminoguanidine, a substance known to inhibit crosslink formation. Second, to examine the extent of glucose incorporation and crosslink formation and the glucosylation-induced structural alterations of the intact glomerular basement membrane, using spectrophotometric and high resolution scanning electron microscopic techniques. Third, to extract three major macromolecular components of the glomerular basement membrane (type IV collagen, laminin, and entactin/nidogen) using denaturing agents, gel permeation and ion-exchange chromatography and to determine possible glucosylation-induced structural alterations using gel electrophoresis, autoradiography and rotary shadowing electron microscopy. Fourth, to examine the ability of the extracted macromolecules to interact with each other and define glucosylation-induced defective interactions using turbidimetry, solid phase binding assays, and rotary shadowing electron microscopy. These studies will broaden our knowledge on the mechanisms underlying structural and functional alterations of the glomerular basement membrane in diabetes and hopefully will lead to proposals aiming at reducing diabetic complications.